Autoinducer compounds and their uses

ABSTRACT

The present invention discloses the autoinducer compounds, such as acyl homoserine lactones, acyl homocysteine lactone, acyl thiolactones, furanones or signal peptides, and their use in animal feed additives and animal feeds to improve animal performance.

FIELD OF THE INVENTION

The present invention relates to autoinducer compounds, otherwise knownas quorum sensing molecules, and to their uses, in particular asadditives to animal feeds for improving animal performance.

BACKGROUND OF THE INVENTION

It has been observed that bacteria in both single culture and mixedcultures are likely to derive significant benefit from the ability toco-ordinate their population dynamics (Shapiro, 1988). ‘Quorum sensing’as this mechanism is known is the ability of bacteria to link geneexpression with population density. Signals produced by the organism areexpressed into their environment and upon critical quorum signallingthen activate a response regulator. This allows single cells to interactwith others of their same and different species. In this manner,bacteria can coordinate their production of defence chemicals,differentiation, reproduction and migration.

Hitherto, by far the most studied applications of the quorum sensingsignal compounds has been in the preparation of diagnostics and thestimulation in vitro of otherwise transiently available antibioticcompounds. It is now recognised that many bacterial species utilise thissignal transduction process by means of a small range of simplemolecules serving as autoinducers of virulence and othercharacteristics. The first molecule to be identified wasN-(3-oxohexanoyl) homoserine lactone (OHHL) as the inducer ofbioluminescence in Vibrio fisheri in 1981 (Eberhard et al, 1981).

In Vibrio species, OHHL production is reliant on density dependent luxgene transcription activated by a protein LuxR. The product Lux1 bindswith LuxR and OHHL to become activated. This then is the general modelprocess for the coordination of various phenotype expression. It wassubsequently found that OHHL was part of a group of compounds, the acylhomoserine lactones (AHLs), many of which (both natural and synthetic)have signalling ability. Other molecules that are known to be quorumsensing signals or autoinducers include various peptides such as‘competence signalling peptide’ in Bacillus subtilis and Streptococcuspneumoniae.

Of the AHLs, N-(3-hydroxybutanoyl) homoserine lactone; N-hexanoylhomoserine lactone; N-butanoyl homoserine lactone; N-(3-oxooctanyl)homoserine lactone; N-octanoyl homoserine lactone; N-(3-oxodecanoyl)homo serine lactone; N-octanoyl homoserine lactone;7,8-cis-N-(3-hydroxytetradecanoyl) homoserine lactone and otheranalogues have also been shown to be active. Other quorum sensingsignals are known to be utilised by certain organisms including3-hydroxypalmitic acid methyl ester.

The AHLs appear to be utilised only in the Gram negative bacteria, whileGram positive bacteria appear to use thiolactone peptide signallingmolecules and other oligopeptides fragments for cell signalling.

The manipulation of the rumen and gut microbiology has hitherto beenaccomplished using antibiotics, including anti-microbials such asvirginiamycin. In the past, many different types of natural andartificial compounds, including sulphonamides, tetracyclines andpenicillin have been used. Their primary function has been to modify therumen microbial populations in such a way as to reduce the undesirablebacteria and favour the beneficial bacteria.

However, there is increasing concern about the long term consequences ofthe use of these compounds in sub-therapeutic concentrations with fearsthat resistance to these drugs may now be widespread.

The complex work done in the rumen reticulum to convert cellulose,hemicellulose and lignins into available energy, while at the same timeproviding the host animal with on-going source of protein, is achievedby a community of bacterial species. This community is intenselycompetitive. Low methane producing rumen systems that are good producersof propionates are better at delivering energy to the animal. Theoptimisation of the rumen to this end has been the enduring target ofdrug and nutritional intervention. The major aim therefore is tomaximise microbial protein production and cellulose/lignin-type compounddegradation, while minimising negative aspects of undesirable microbialgrowth. The organisms that consume or degrade protein and increase(energy consuming) methane production are themselves non-mutualistic intheir relationship with the host or deprive the animal of availablestarch and are therefore considered deleterious to optimal rumenfermentation.

The difficulty in using feed additives or other bioactive treatments isthat of specificity as many substances have multifold effects on themicrobial community. Reduction of proteolysis and deamination activityis partly responsible for increased performance of the animal. Thecontrol of the microbial population can also positively influence theproduction of volatile fatty acids and reduce methane production. Thecombined manipulation of these parameters results in improved animalperformance, sometimes by very substantial margins.

WO97/27851 (The Johns-Hopkins University) discloses that the growth ofMycobacteria can be inhibited through the administration of homoserineor a homoserine lactone. The application suggests that these compoundsare used in the diagnosis and treatment of M. tuberculosis infection inhumans.

U.S. Pat. No. 5,591,872 (University of Iowa) discloses thatN-(3-oxododecanoyl) homoserine lactone is an autoinducer which regulatesgene expression in Pseudomonas aeruginosa and says that analogs orinhibitors of this autoinducer can be used in treating or preventinginfection by this microorganism.

WO01/74801 (University of Nottingham) discloses a family of N-acylhomoserine lactones and their use as immunosuppressants.

SUMMARY OF THE INVENTION

Broadly, the present invention relates to the synthesis and use ofquorum sensing molecules or autoinducers in animal diets, particularlyin feedstuffs for ruminant and monogastric animals. Recent legislativechanges have made the use of most antibiotics illegal in many Westerncountries and the new solutions have been sought to improve fermentationusing more natural antimicrobials. However, many of these naturalproducts have unreliable efficacy records and are themselves likely tobe scrutinised by the regulatory authorities. Therefore, a completelynew approach to the optimisation of fermentation is desirable. The useof autoinducer compounds or quorum sensing signals in animal feed asdisclosed in the present invention provides a novel way of controllingthe dynamics of the rumen flora using the very compounds the naturalmicrobial population produce themselves.

In the present invention, the terms ‘quorum sensing molecule’ and‘autoinducer compound’ are used interchangeably. Examples of thesecompounds are set out below.

In a first aspect, the present invention provides an animal feedadditive comprising one or more autoinducer compounds. Optionally, theautoinducer compound is admixed with an inert carrier to bulk it upprior to mixing with animal feed ingredients, provided as a solution foradminstration as a drench or for spraying onto animal feed, orformulated in tablet form, again with an inert carrier. Examples ofinert carriers include silica-talc and water.

In a further aspect, the present invention provides an animal feedcomprising an animal feed component and one or more autoinducercompounds. Examples of animal feed components include one or acombination of proteins, sugars, fats and fibre. Typically, animal feedcomponents are derived from cereals and other plant material.

In a further aspect, the present invention provides a non-therapeuticmethod of improving animal performance comprising administering anautoinducer compound to the animal.

In a further aspect, the present invention provides the use of anautoinducer compound for administering to an animal for the purpose ofimproving animal performance.

Examples of autoinducer compound or compounds include acyl homoserinelactones, acyl homocysteine lactones, acyl thiolactones, signal peptidesand signal furanones and quinolines, such as2-heptyl-3-hydroxy-4-quinoline. Preferably the acyl lactones are C₁₋₂₀acyl lactones.

Examples of preferred acyl homoserine lactones include compounds such asN-oxobutanoyl homoserine lactone, N-oxopentanoyl homoserine lactone,N-oxohexanoyl homoserine lactone, N-oxoheptanoyl homoserine lactone,N-oxooctanoyl homoserine lactone, N-oxononanoyl homoserine lactone,N-oxodecanoyl homoserine lactone, N-butanoyl homoserine lactone,N-pentanoyl homoserine lactone, N-hexanoyl homoserine lactone,N-heptanoyl homoserine lactone, N-octanoyl homoserine lactone,N-nonanoyl homoserine lactone, N-decanoyl homoserine lactone, and7,8-cis-N-(3-hydroxytetradecanoyl) homoserine lactone. N-oxoacylhomoserine lactones are preferably N-(3-oxoacyl) homoserine lactonessuch as. The synthesis of further examples of acyl homoserine lactonesis described in WO01/74801.

Examples of preferred acyl homocysteine lactones include compounds suchas N-oxobutanoyl homocysteine lactone, N-oxopentanoyl homocysteinelactone, N-oxohexanoyl homocysteine lactone, N-oxoheptanoyl homocysteinelactone, N-oxooctanoyl homocysteine lactone, N-oxononanoyl homocysteinelactone, N-oxodecanoyl homocysteine lactone, N-butanoyl homocysteinelactone, N-pentanoyl homocysteine lactone, N-hexanoyl homocysteinelactone, N-heptanoyl homocysteine lactone, N-octanoyl homocysteinelactone, N-nonanoyl homocysteine lactone, and N-decanoyl homocysteinelactone.

Examples of preferred acyl thiolactones include compounds such asN-oxobutanoyl thiolactone, N-oxopentanoyl thiolactone, N-oxohexanoylthiolactone, N-oxoheptanoyl thiolactone, N-oxooctanoyl thiolactone,N-oxononanoyl thiolactone, N-oxodecanoyl thiolactone, N-butanoylthiolactone, N-pentanoyl thiolactone, N-hexanoyl thiolactone,N-heptanoyl thiolactone, N-octanoyl thiolactone, N-nonanoyl thiolactone,and N-decanoyl thiolactone.

In other embodiments, the autoinducer compounds are represented by oneof the formulae:

wherein X and Y are independently selected from O, S or NH and Z is asubstituted or unsubstituted C₁ to C₂₀ acyl chain. The acyl chain may bebranched or unbranched, unsaturated, partially saturated or saturated.Examples of acyl chain substituents include keto, hydroxy, alkenyl orphenyl substituents. The autoinducer compound may be partially orcompletely halogenated.

In embodiments in which the autoinducer compound is chiral, it may bepresent as a single enantiomer or any mixture of optical isomers.

In addition to the autoinducer compound, the animal feed additive oranimal feed may contain other ingredients such as antibiotics, such asTylsine, tetracycline, gentamycin, bactracin-methylene-disalicylate andvalnemulin, or coccidiostats such as salinomycin.

In the present invention, the term ‘improving animal performance’includes improving animal growth rates, improving animal weight at agiven age, improving feed conversion ratio, improving the yield orquality of a product produced by or derived from the animal (e.g. meat(e.g. from livestock, poultry or fish), milk from lactating livestock oreggs from poultry), all of these being defined in relation to controlanimals who are untreated with the autoinducer compound. Thesecomparisons can be readily made by those skilled in the art, e.g. feedconversion ratio can be calculated on the basis of feed consumed/totalweight of animals in a sample.

While not wishing to be bound by any specific theory, the applicantsbelieve that the inclusion of autoinducer compounds in animal diet has abeneficial effect on the populations of bacteria in the digestive tractof animals. This effect may be in the regulation of gene expression inanimal gut bacteria in vivo, in the promotion of surfactant productionby gut flora as surfactants can aid in the emulsification of the fat orlipid content of feed making it more readily available to the animal, inthe promotion of virulence in specific rumen fluid bacteria or in theproduction of antibiotics by monogastric gut bacteria.

The autoinducer compounds may be administered to animals such as birds,livestock, marine animals or domestic or companion animals. Examples ofthese animals include poultry, cattle, swine, sheep, rabbits, horses,dogs and cats, and fish, e.g. in aquatic farming.

Preferably, the autoinducer is administered directly or indirectly to ananimal at a dose equivalent to 1 to 100,000 nanomoles per tonne of feed,more preferably 100 to 10,000 nanomoles per tonne of feed, and mostpreferably about 1,000 nanomoles per tonne of food.

The autoinducer compound can be provided to the animal by a wide rangeof routes. As an animal feed additive it can be formulated as a drypowder (e.g. for mixing with animal feeds), a liquid (e.g. for sprayingonto animal feeds or animal drinking water), or formulated for directapplication into animal feeds. Alternatively, the autoinducer compoundcan be supplied premixed with an animal feed or administered directly tothe animal as a supplement. Additionally or alternatively, a compositioncomprising the autoinducer compound may be in the form of a capsule ortablet, formulated as a drench or be in the form of a bolus for theingestion by an animal. In these embodiments, the autoinducer compoundmay be formulated by admixing it with an inert carrier, e.g. a solventsuch as water or a solid carrier such as silica talc, to making dosingeasier in the field.

In a further aspect, the present invention provided a method of makingan animal feed, the method comprising mixing one or more animal feedcomponents with one or more autoinducer compounds. The method maycomprise additional steps in the processing of the feed, e.g.pellitisation.

In some embodiments, the autoinducer compounds may be made by syntheticchemistry techniques. Alternatively, the compounds may be derived fromextracts or concentrates of plants, algal, fungal or bacterial material.As a further alternative, the autoinducer compounds can be derived fromgenetically modified organisms that over-express the autoinducercompounds, either naturally or because they have been transformed.Examples of transformed organisms include bacteria or plant cellstransformed with nucleic acid encoding autoinducer compounds such as anacyl homoserine, homocysteine or thiolactone lactone synthase gene orgene cluster or with nucleic acid encoding a signal peptide. Thetransformed host cells may then be induced to express the autoinducercompound which optionally may then be purified from cell culture andformulated as described above. Alternatively, an animal feed or animalfeed additive may be directly made from the bacteria or plant cells,e.g. by making an animal feed from a plant which has been geneticallyengineered to over-express one or more of the autoinducer compounds.

In a further aspect, the present invention provides a method ofpreparing acyl homoserine lactone compounds, the method comprisingrefluxing amino butyrolactone with an acetate compound to produce theacyl homoserine lactone. In this method, preferably the solvent employedis toluene, xylene or ethylbenzene, and more preferably the solvent istoluene. Preferred reactions conditions are refluxing the reactionmixture under atmospheric pressure.

Preferably, the acetate compound is ethyl butanoate, ethyl pentanoate,ethyl hexanoate, ethyl heptanoate, ethyl octanoate, ethyl nonanoate,ethyl decanoate, ethyl 3-oxobutanoate, ethyl 3-oxopentanoate, ethyl3-oxohexanoate, ethyl 3-oxoheptanoate, ethyl 3-oxooctanoate, ethyl3-oxononanoate or ethyl 3-oxodecanoate.

The method may comprise the additional step of purifying the acylhomserine lactone produced in the reaction. In one embodiment, this canbe achieved by evaporating the product, redissolving in 5% methanol indichloromethane and purified by column chromatography.

By way of example and not limitation, embodiments of the presentinvention will now be described in more detail.

DETAILED DESCRIPTION EXAMPLE 1 Effect of Dietary Lactones on theDigestion of Grass Silage by Cultures of Rumen Fluid from HealthyFistulated Grass Fed Cows

Autoinducer compounds such as acyl homoserine lactones can havesignificant effects on cultures of bacteria. For example, they may beused to induce expression of antibiotics and extracellular enzymes.Hexanoyl homoserine lactone (OHHL) is the signal molecule for antibioticproduction in Chromobacterium violaceum; butanoyl homoserine lactonetriggers various phenotypes in Pseudomonas aeruginosa including theproduction of various enzymes and lectins. OHHL is known to havedifferent effects on different species. For example, in Erwiniastewartii it induces production of exopolysaccharides, while in Vibriospecies it promotes bioluminescence. Thus, in a complex mixed culturecomprising many species including ruminobacter sp., prevotella sp.,ruminococcus sp., it is hard to predict the specific phenotypes thatwill be induced by even the introduction of just one autoinducercompound. However. the global effect on rumen mixed culture fermentationcan be measured in terms of the efficiency of digestion of forage. Inthe following example, an in vitro model of animal rumen efficiencyreveals the net effect on the digestion of forage using fresh rumenfluid. A control sample treated with water and four test samples treatedwith using nanomolar concentrations of OHHL are examined.

Rumen fluid was collected from a healthy fistulated grass fed cow andimmediately dispensed into 75 ml bottles. These bottles were kept at 37°C. Into each of these cultures approximately 1 g of pre-weighed,pre-dried grass silage was suspended in sachets made from nylon gauze.Signal AHL [OHHL] was introduced at this point. in concentrations thatprovided final concentrations in the rumen fluid of 0, 200, 500 and 1000nanomoles. These were then incubated for 10 hours. The forage sampleswere then removed and re-dried and re-weighed. Each treatment wasconducted in triplicate.

The results of the experiments showed that increasing the amount of OHHLpresent increased the mean percentage loss of weight for each treatment,indicating that the presence of autoinducer compounds leads to improvedefficiency in the digestion of animal feed.

When added to feed the inclusion level should be increased from theabove levels to allow for losses during the feed extrusion processes.For this reason, a typical inclusion rate of 5-5000 nanomoles is usuallysufficient.

EXAMPLE 2 Synthesis of an Autoinducer Compound

Very few of the AHL compounds are available commercially and synthesisprotocols in the literature involve many steps and low yields. It wasthus important that an inexpensive synthetic route was perfected thatcan serve as the model route for all of the AHL compounds. OHHL which ispure by NMR was prepared as follows.

To a stirred mixture of α-amino-γ-butyrolactone (1.0 eq) in toluene (˜5ml/per mmol) was added triethylamine (1.0 eq) dropwise. The mixture wasthen stirred for 10 minutes. Ethylbutyryl acetate (1.0 eq) was addeddropwise and the mixture refluxed for 2 hours. The mixture was allowedto cool and was then filtered and evaporated. Column chromatography with5% methanol in dichloromethane gives the compound in >30% overall yield.

NMR analysis confirmed the presence of OHHL: Probe head 5 mm H¹; AQ1.9923444 sec; TE 300.0K 1 D NMR plot parameters: cx 40.0 cm; F1P 10.5ppm; F2P—0.500 ppm; 110.03576 Hz/cm

NMR δvalues for OHHL: 7.609 ppm; 4.525 ppm; 4.412 ppm; 4.20 ppm; 3.402ppm; 2.677 ppm; 2.45 ppm; 2.16 ppm 1.56 ppm 0.844 ppm.

It is known that AHL's are present in the rumen (Erickson et al, 2000).Reverse phase thin layer chromatography of rumen fluid revealed thepresence of ‘multifold’ signals.

It is thus clear that many of the bacterial species already derivecompetitive benefit from quorum sensing mechanisms.

As described, synthetic signal compounds may be introduced to the rumenin small doses through the animal feed to improve rumen efficiency andtherefore improve animal performance. It is known that multiple lactonesignals regulate virulence determinants in species such as Pseudomonassp. Quinolones such as 2-heptyl-3-hydroxy-4-quinoline are also activesignal molecules and may also be utilised to improve animal nutritionand health.

Combinations of the AHL compounds may be used to further manipulaterumen events, but the precise formulations into the feedstuff willnecessarily depend upon the species and feedstuff concerned. Signaleavesdropping, where an optimised cow rumen is analysed for signals thatare then artificially reproduced and then introduced is another option.Similarly, in monogastrics, quorum sensing signals may be utilised tostimulate the production of antibiotics by beneficial gut flora.Additionally, other beneficial bacterial products such as enzymes andsurfactants may also be induced using this technology.

Inactive analogues of signal molecules can be used to competitivelyinterfere with the signalling process (‘signal jamming’). In thisscenario, transcription of, for example, virulence genes of deleteriousgut bacteria, can be forestalled and pathogenic damage mitigated. Thesubsequent improvement in animal health will thus contribute to overallanimal performance. It may be possible to harvest signal molecules fromcultures in in vitro fermentation and signal peptides (typically thequorum sensing signals for Gram positive bacteria) could be prepared bygenetic manipulation, for example to allow over-expression of peptidessuch as the oligopeptides used by Enterococcus faecilis.

EXAMPLE 3 Effect of Dietary Lactones on Growth Performance and Mortalityof Broiler Chickens Raised in Floor Pens

Materials and Methods

This experiment examined the effect of N-(3-oxohexanoyl)-L-homoserinelactone (OHHL) (CAS #: 143537626, molecular formula : C₁₀H₁₅NO₄,molecular weight: 213) on the growth performance and mortality ofbroiler chickens.

A stock solution of 1 mM OHHL (0.213 g/L) was prepared as follows.Approximately 50% of the required volume of distilled water was warmedto approximately 30-40° C. and used to dissolve the required amount ofOHHL powder. The solution was made up to volume using distilled waterstored at room temperature. OHHL solution (0.213 g/L) was applied totreated crumbled feed at a rate of 3 kg per tonne. Control feed wastreated with distilled water at a rate of 3 kg per tonne. The OHHLsolution was stored for less than 2 days prior to application to feed.

The antibiotic BMD®110 was used as a positive control in this study. Theactive ingredient is bacitracin methylene disalicyclate. The productcontains 110 g of bacitracin activity per kg and is approved forprevention of necrotic enteritis in broiler chickens when given at adose of 55 ppm in feed (500 g BMD®110/tonne feed).

Coxistac® 6% premix was used as an ionophore in all study diets as anaid in the prevention of coccidiosis. The product contained 60 gsalinomycin per kg and was administered at a dose of 60 ppm in feed (1kg Coxistac® 6% premix per tonne feed).

The experiment lasted 35 days with the day of placement of broilerchicks considered as day zero. A total of 1,200 male day-old broilerchickens (Cobb×Cobb) were assigned to treatment on day 0.Birds werevaccinated for Marek's disease at the hatchery. Twenty-four pens, eachproviding 45 square feet of floor space, were assigned to treatmentgroups. Each pen had a concrete floor and a 12-inch high concretebarrier at the front and back. Adjacent pens were separated by a solid12-inch high plastic barrier at bird level. A welded wire fence with1-inch square openings was located on top of all barriers. Each pen waspermanently identified by number and contained 50 birds on day zero.Each pen contained four nipple-type drinkers which provided cleandrinking water ad libitum. Dry feed was provided ad libitum in tube-typefeeders (one per pen) of 20 kg capacity.

The barn was heated by five natural gas heaters which were equallyspaced and positioned to warm incoming air at the north wall of thebuilding. Air was exhausted by fans located on the south-facing wall ofthe building. Lighting program, barn temperature, and other managementpractices were typical of commercial broiler chicken producers in NorthAmerica. Birds that were moribund and unable to reach food or water wereculled and euthanised by carbon dioxide gas.

Bodyweight, pen number and date of death were recorded for each birdthat was culled or found dead. Mortalities were submitted to thepathologist to determine the apparent cause of death.

A randomised complete block design was used to study the main andinteractive-effects of OHHL (O and 0.639 g/tonne) and dietary antibiotic(0 and 55ppm BMD®110)in a 2×2 factorial arrangement. Dietary treatmentswere as follows:

Treatment OHHL, BMD ® 110, code g/tonne g/tonne A 0 0 B 0.639 0 C 0 500D 0.639 500 *All diets contained 60 ppm salinomycin (Coxistac ®)

There were four pens per block and six replicate blocks for a total of24 pens.

The feeding program was used in the study used a starter feed type ondays 0 to 20 and a grower feed type on days 21 to 35. Diet formulationwas representative of commercial diets in North America.

A starter diets using a basal mix of starter diet containing either 0 or55 ppm BMD was manufactured, pelleted, and crumbled. Bagged starter feedwas treated with either distilled water (0 g OHHL per L) or OHHLsolution (0.213 g OHHL per L) using a horizontal double ribbon mixer of100 kg capacity. Distilled water or OHHL solution (0.213 g per L) wereapplied to crumbled feed at a rate of 3 kg per tonne feed. Grower dietswere manufactured as described above for starters.

Feed sampling and assay: a minimum of 10 representative samples weretaken from each batch of crumbled basal starter and grower feed. The 10samples were composited and divided into two samples for nutrient assayand retainer sample, respectively. A representative composite sample ofeach control and OHHL-treated feed were taken. Duplicate samples(analytical and retainer) were stored frozen at −20° C. forretrospective OHHL assay. One sample of each crumbled basal feed wasanalysed for dry matter, crude protein, calcium, phosphorus andmanganese.

The data collected consisted of:

-   1. Bodyweight on days 0, 21, and 35.-   2. Amounts of each feed (starter and grower) consumed.-   3. Bodyweight and date of death for birds which were culled or died.-   4. Feed conversion ratio was calculated on a pen basis as feed    consumed/[total weight of live birds+total weight of dead and culled    birds+total weight of sacrificed birds].-   5. Average bodyweight per pen was calculated as total weight of live    birds at time of weighing/number of live birds at time of weighing.-   6. Daily feed intake per bird was calculated on a pen basis for the    starter and grower periods as total feed consumed divided by number    of live bird days in the specified period.-   7. Apparent cause of death was recorded for all birds that died or    were culled.-   8. Birds were observed on a flock basis at least once daily and    observations recorded.-   9. Cause of death.    Statistical Analysis

The pen was the experimental unit for statistical analysis. Mortalitydata was transformed using an arcsine transformation (Steel and Torrie,1980) prior to analysis of variance. All data were analysed by analysisof variance using the following model:

Degrees of Source freedom OHHL 1 Antibiotic 1 OHHL × Antibiotic 1 Block5 Residual error 15  Total 23 

Means were compared using an appropriate multiple range test (Steel andTorrie, Principles and procedures of statistics, a biometrical approach.McGraw Hill Book Co., NY., 1980).

Results and Discussion

Dietary administration of OHHL significantly improved (P=0.024) Day 21bodyweight of broiler chickens (Table 1). There was no significanteffect of dietary BMD on bodyweight.

Administration of OHHL improved feed efficiency of broilers on Day 21(P=0.012) and for the overall Day 0-35 period (P=0.055). Dietary BMDalso improved feed efficiency for the Day 21-35 period (P<0.001) and theoverall growth period (P=0.014).

There was a significant OHHL×BMD interaction effect for feed efficiencyduring the starter period. However, this is attributable to a poor feedefficiency in birds that received only BMD in the starter period (feedefficiency=1.422). The feed efficiency response to OHHL in combinationwith BMD was slightly greater than the response to OHHL alone.

Morbidity and Mortality

Old litter was used in the present study in an attempt to create asubstantial disease challenge. However, overall mortality was very lowin comparison to commercial norms of 4 to 5%. In the absence of BMD,OHHL reduced mortality from 2.0% to 1.7%. In the presence of BMD, OHHLreduced mortality from 3.3% to 2.7% (Table 2). These numeric changes inmortality are not statistically significant but do provide preliminaryevidence that continuous administration of OHHL did not have an adverseeffect on bird survival.

The final bodyweights and feed efficiency data also suggest excellentgrowth performance and minimal flock morbidity.

All mortalities were necropsied and there was no evidence of unusual oradverse drug effects in the study.

Conclusions

Continuous administration of OHHL to broiler chickens improved Day 21bodyweight (P=0.024) and overall feed efficiency (P=0.055).

Mortality of OHHL-treated broilers was numerically lower than non-OHHLtreated controls both in the presence and absence of dietary BMD.

There was no evidence of any adverse effect of OHHL on bird health.

EXAMPLE 4 Effect of Dietary Lactones on Rumen Dry Matter Disappearancein Sheep

Materials and Methods

This experiment examined the effect of N-(3-oxohexanoyl)-L-homoserinelactone (OHHL) (CAS#: 143537626, molecular formula : C₁₀H₁₅NO₄,molecular weight: 213) on rumen dry matter disappearance in vivo insheep.

A stock solution of OHHL (0.639 gram/L) was prepared as follows.Approximately 50% of the required volume of distilled water was warmedto approximately 30-40° C. and used to dissolve the required amount ofOHHL powder. The volumetric flask was made up to volume using distilledwater stored at room temperature. OHHL solution was applied to pelletedsheep ration at a rate of 3 kg per tonne of feed. Control feed wastreated with 3 kg of distilled water per tonne.

A batch mixer and appropriate spraying device were used to ensureuniform application of liquid to the feed. Control feed was manufacturedfirst to avoid cross contamination with OHHL. It was anticipated thatsheep ration would comprise approximately one third of total dry matterintake of study animals based on an estimated dry matter intake of 2% ofbodyweight.

Initial attempts to administer OHHL by application of an aqueoussolution to the outside of pelleted feed were modified as the sheepdecreased intake of treated feed after a few days. Instead, OHHL wasadministered as an oral drench twice daily commencing on the afternoonof Day 11 of each period.

Animals were individually penned to minimize the potential for damage tocannulae and to permit individual feeding. Fresh drinking water wasprovided ad libitum.

A restricted quantity of pelleted ration was provided at a rate ofapproximately 0.5 kg/day (0.25 kg in the a.m. and 0.25 kg in the p.m.).Access to hay was restricted as needed to help ensure that sheep rationwas consumed.

On Days 12 and 13, pelleted ration (0.25 kg/animal) was issued toanimals approximately 1 hour prior to introduction of bags into therumen and again following removal of the 8-hour bag from each animal.

One fresh sample of corn silage was dried to constant weight and allowedto cool to room temperature. A representative sub-sample was taken fordry matter determination. The sample was ground to pass a 1 mm screen,mixed and sampled for dry matter assay. The remaining sample was storedfor in situ determination of dry matter disappearance from the rumen ofcannulated sheep.

Ankom rumen sampling bags were used in the study. Each bag wasapproximately 5 cm×10 cm, suitable for a one-gram sample. Pore size was53±10 microns. Dried, ground corn silage was weighed (1.00±0.01 grams)into bags and sealed. A set of four bags was prepared per animal per dayand these were attached to a string to facilitate placement in andremoval from the rumen. A fifth bag served as a blank for each set offour bags. The blank was not placed in the rumen but was washed,processed and dried.

A rumen cannula was surgically placed in each of five mature(approximately 3-year old) ewes. Following recovery from surgery, fourof these animals were selected for use in the study. The fifth animalserved as a reserve for use in the event of post-surgical complicationsin a study animal.

Dry matter disappearance was measured by removing bags from the rumen at4, 8, 12 and 24 hours and washed under cold running tap water togetherwith a corresponding blank bag. Bags were then dried to constant weight.Measurement of dry matter disappearance was completed for each animalcommencing on the morning of Day 12 and Day 13 of each period.

A Latin Square Design was used to study the effects of two treatments:

A, Control: 0 gram OHHL per tonne ration.

B: Treated with the equivalent of 1.917 gram OHHL per tonne ration.

Each period was of 14 days duration. A total of four study animals wereblocked based on bodyweight (2 blocks). Animals within block wererandomly assigned to Sequence 1 or 2:

Sequence 1 Sequence 2 Period 1 Treatment A Treatment B Period 2Treatment B Treatment AStatistical Analysis

Data were analysed by a multiple regression analysis that includedeffects of treatment, animal, period, study day and hour.

Animal Health

In Period 1, prior to measurement of in situ dry matter disappearance,one OHHL-treated sheep was removed from study due to poor appetite andwas replaced with a reserve animal. The removed animal was euthanized,necropsied and found to have a liver abscess developed prior to theexperiment.

Dry Matter Disappearance

There was a highly significant (P<0.0001) effect of rumen incubationtime on dry matter disappearance as expected. After 4 and 24 hours ofincubation, approximately 50% and 75% of dry matter had disappeared fromAnkom bags (Table 1). Dry matter disappearance was measured on twoconsecutive days in each period but there was no significant (P=0.97)effect of day on this variable.

Treatment means are summarized in Tables 3 and 4.OHHL improved(P=0.105,Table 2) mean dry matter disappearance by 1.77 percentageunits. The magnitude of the response varied considerably with incubationtimes but this is largely a reflection of the variation inherent in suchmeasurements.

Conclusions

The experiment shows that administration of OHHL improved (P=0.105) drymatter disapearance of corn silage in the rumen of sheep.

TABLE 1 OHHL and BMD effects on bodyweight and feed intake of broilerchickens (1 = no; 2 = yes) (1 = no; 2 = yes) Average bodyweight, kgDaily Feed intake OHHL BMD Day 0 Day 21 Day 35 Day 0-21 Day 21-35 Day0-35 1 0.0416 0.762 1.922 0.048 0.147 0.087 2 0.0415 0.785 1.937 0.0480.147 0.087 Significance NS 0.024 NS NS NS 1 0.0416 0.781 1.922 0.0480.147 0.087 2 0.0415 0.766 1.937 0.048 0.147 0.087 Significance NS NS NSNS NS NS 1 1 0.0415 0.767 1.910 0.047 0.148 0.087 2 1 0.0417 0.796 1.9340.049 0.147 0.088 1 2 0.0416 0.758 1.934 0.049 0.147 0.087 2 2 0.04130.774 1.940 0.047 0.147 0.087

TABLE 2 OHHL and BMD effects on feed efficiency of broiler chickens (1 =no; 2 = yes) (1 = no; 2 = yes) Feed Conversion Mortality OHHL BMD Day0-21 Day 21-35 Day 0-35 Day 0-21 Day 21-35 Day 0-35 1 1.397 1.784 1.6342.0 0.7 2.7 2 1.353 1.794 1.620 1.3 0.8 2.2 Significance 0.012 NS 0.055NS NS NS 1 1.364 1.816 1.637 1.2 0.7 1.8 2 1.386 1.762 1.617 2.2 0.8 3.0Significance 0.071 0.000 0.014 NS NS NS Significance (OHHL *BMD) 0.0250.094 NS NS NS NS 1 1 1.372 1.820 1.644 1.3 0.7 2.0 2 1 1.357 1.8121.629 1.0 0.7 1.7 1 2 1.422 1.749 1.624 2.7 0.7 3.3 2 2 1.350 1.7751.610 1.7 1.0 2.7 NS, P > 0.10

TABLE 3 OHHL effect on dry matter disappearance, % Hours Treatment 4 812 24 Mean Control 48.8 58.4 62.3 74.4 61.0 OHHL 53.0 57.8 65.7 74.562.7 OHHL − Control 4.16 −0.60 3.36 0.15 1.77

TABLE 4 Significance of independent variables for prediction of DMdisappearance Variable P value Treatment 0.105 Sheep 0.000 Period 0.023Study Day 0.974 Incubation time, hour 0.000

References

The references mentioned herein are all expressly incorporated byreference.

-   1. Shapiro, Bacteria as multicellular organisms, Scientific American    246: 82-89, 1988.-   2. Eberhard et al, Structural identification of autoinducer of    photobacterium fisheri luciferase, Biochemistry, 20: 2444-2449,    1981.-   3. Reprod. Nutr. Dev., 40: 189-202, 2000.-   4. Erickson et al, Reprod. Nutr. Dev., 189-202, 2000.-   5. WO01/74801 (University of Nottingham)

1. In an animal feed providing improved animal performance, theimprovement which comprises the incorporation in said animal feed of atleast one autoinducer compound, in an amount effective to improveefficiency in the digestion of an animal receiving said animal feed,thereby improving animal performance, said autoinducer compound being anacyl homoserine lactone.
 2. The animal feed of claim 1, wherein saidanimal feed comprises at least one of proteins, sugars, fats or fibre.3. The animal feed of claim 1, wherein said animal feed is derived fromcereals or other plant material.
 4. The animal feed of claim 1, whereinthe autoinducer compound is selected from a group consisting ofN-oxobutanoyl homoserine lactone, N-oxopentanoyl homoserine lactone,N-oxohexanoyl homoserine lactone, N-oxoheptanoyl homoserine lactone,N-oxooctanoyl homoserine lactone, N-oxononanoyl homoserine lactone,N-oxodecanoyl homoserine lactone, N-butanoyl homoserine lactone,N-pentanoyl homoserine lactone, N-hexanoyl homoserine lactone,N-heptanoyl homoserine lactone, N-octanoyl homoserine lactone,N-nonanoyl homoserine lactone, and N-decanoyl homoserine lactone.
 5. Theanimal feed of claim 1, wherein said autoinducer compound is representedby one of the formulae:

wherein X and Y are —O— and Z is a substituted or unsubstituted C₁ toC₂₀ acyl chain.
 6. A non-therapeutic method of improving animalperformance comprising administering to an animal selected from thegroup consisting of ruminant and monogastric animals an animal feedcomprising at least one autoinducer compound, in an amount effective toimprove efficiency in the digestion of an animal receiving said animalfeed, thereby improving animal performance, said autoinducer compoundbeing an acyl homoserine lactone.
 7. The method of claim 6, whereinimproving animal performance comprises improving animal growth rate,improving animal weight at a given age, improving feed conversion ratio,improving the yield or quality of a product produced by or derived fromthe animal.
 8. The method of claim 7, wherein the product produced orderived from the animal is eggs, milk or meat.
 9. The method of claim 6,wherein the animal feed is administered to birds, livestock, domestic orcompanion animals or marine animals.
 10. The method of claim 9, whereinthe animals are poultry, cattle, swine, sheep, rabbits, horses, dogs,cats or fish.
 11. The method of claim 6, wherein the autoinducer isadministered to the animal at a dose equivalent to 1 to 100,000nanomoles per tonne of feed.
 12. The method of claim 11, wherein theautoinducer is administered to the animal at a dose equivalent 100 to10,000 nanomoles per tonne of feed.
 13. The method of claim 6, whereinthe autoinducer compound is formulated as a powder, a liquid, a capsuleor tablet, a drench or a bolus.
 14. The method of claim 6, wherein theautoinducer compound is obtained from plant, algal, fungal or bacterialmaterial.
 15. A method of making an improved animal feed, the methodcomprising the steps of: a) providing an animal feed; b) mixing withsaid animal feed at least one autoinducer compound, said autoinducercompound being an acyl homoserine lactone; and c) optionally pelletizingthe mixture obtained from step b, together with an inert carrier.